Paxillin stimulating compositions and cosmetic uses thereof

ABSTRACT

Cosmetic compositions comprising one or more paxillin stimulators and methods of using such compositions to impart anti-aging benefits to the skin are disclosed. The paxillin stimulators and combinations thereof are believed to have modulatory activity against at least one biochemical pathway implicated in skin aging.

CROSS-REFERENCE TO A RELATED APPLICATION

The present application is a divisional application of parentapplication U.S. Ser. No. 12/966,098, filed Dec. 13, 2010, which in turnclaims the benefit of provisional application U.S. Ser. Nos. 61/290,720filed Dec. 29, 2009 and 61/289,038 filed Dec. 22, 2009, all of which areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to compositions for topicalapplication to the skin which comprise at least one paxillin stimulatorand the use of such compositions to provide benefits to the skin, inparticular, to provide anti-aging benefits to human skin.

BACKGROUND OF THE INVENTION

Consumers continually seek to improve the appearance of their skin andin particular to reduce visible signs of skin aging. Unwanted signsinclude lines and wrinkles, skin sagging or atrophy, and loss ofsuppleness, and there remains a need for products that combat such signsof aging and, more generally, that provide anti-aging and/oranti-wrinkle effects.

Recent studies have revealed that dermal fibroblasts undergo morphologychanges and cell body collapse in both chronically and photo-aged skin.See, e.g., Varani et al., 2004. J. Invest. Dermatol. 122:1471-9; andVarani et al., 2006. Am. J. Pathol. 168: 1861-8. Such alterations canlead to coarse, rough, and wrinkled appearance, which arecharacteristics of aged skin. Further studies suggest that collagendegradation along with altered integrin and focal adhesion molecules arefactors contributing to the loss of a functional dermal collagen matrix,with the consequence of cell body collapse due to a loss of mechanicaltension between fibroblasts and the matrix. See, e.g., Fisher et al.,2008. Arch Dermatol. 144: 666-72.

Paxillin is an important adaptor protein that mediates transmembraneintegrins and growth factor signaling. It transduces messages from theextracellular matrix, recruits other focal adhesion molecules to formcomplexes, and activates intracellular cytoskeleton assembly. Brown etal., 2004. Physiol Rev. 84:1315-39. This process is important for celladhesion and migration, as well as muscle contraction. Paxillin-mediatedsignaling also affects long-term changes in gene expression, cellproliferation, and extracellular matrix organization, which is importantto wound repair and tissue regeneration.

Paxillin exists in higher eukaryotes as three isoforms. Paxillin a isthe principle, ubiquitously expressed isoform, expressed in most adulthuman tissues other than brain; paxillin β- and γ-isoforms arerestrictively expressed. A fourth isoform, paxillin δ, is found mainlyin epithelial cells. The paxillin proteins are comprised of multipleprotein-binding motifs, corresponding to multiple protein-proteininteraction and protein recognition sites, with many phosphorylationsites dispersed throughout the molecule. Paxillin binding partners rangefrom structural actin-binding proteins, such as vinculin, to signalingmolecules such as focal adhesion kinase (FAK) and integrin-linked kinase(ILK). Paxillin is phosphorylated at the multiple tyrosine, serine, andthreonine sites in response, e.g., to cell adhesion and/or varioussoluble growth factors and cytokines and is thought to be at thesignaling crossroads of cell adhesion and growth factor modulation undernormal conditions.

Following exposure to oxidative stress, abnormalities have been observedin paxillin phosphorylation and cytoskeletal organization in culturedcells. Hao et al., 2006. Free radical Biol Med. 41: 302-10; and Zhou etal., 1999. J Cell Physiol. 180: 182-9. Also, altered levels and alteredlocalization of various focal adhesion proteins have been observed insenescent cells in vitro. Nishio et al., 2005. Histochem cell biol.123:263-73. Nonetheless, no direct relation has been implicated betweenpaxillin expression and visible signs of aging in human skin.

It is therefore an object of the invention to provide new approaches forcombating signs of skin aging through paxillin-mediated pathways. It isa further object of the invention to provide new compositions andmethods directed thereto. It is a still further object of the inventionto improve the overall appearance of skin, including treating,reversing, and/or preventing signs of aging, using cosmetic compositionscomprising effective amounts of one or more compounds that affectpaxillin production.

The foregoing discussion is presented solely to provide a betterunderstanding of nature of the problems confronting the art and shouldnot be construed in any way as an admission as to prior art nor shouldthe citation of any reference herein be construed as an admission thatsuch reference constitutes “prior art” to the instant application.

SUMMARY OF THE INVENTION

In accordance with the foregoing objectives and others, it hassurprisingly been found that disruption of normal paxillin production inhuman skin fibroblasts directly leads to changes in cell shape,indicating an essential role of paxillin in maintaining optimum cellmorphology and optimum cell health. It further has surprisingly beenfound that paxillin levels in human skin cells can be stimulated bytopical application of compositions comprising one or more paxillinstimulators, indicating a new approach to combat signs of skin aging.

In one aspect of the invention, cosmetic compositions are provided forimproving the aesthetic appearance of skin comprising, in a cosmeticallyacceptable vehicle, an effective amount of at least one paxillinstimulator. The paxillin stimulator may be selected from the groupconsisting of pyridone-fused azabicyclic compounds having the structureof formulae IV or V; Jasminum sambac extract; Coccinia grandis extract;Eliptica prostrata Linn. extract; Clitoria ternatea Linn. extract;Ozothamnus obcordatus extract; Erythrina flabelliformis extract;Lonchocarpus capassa extract; Sophora tomentosa extract; Trifoliumhybridum extract; Eremophila mitchellii extract; Kunzea ambigua extract;Tanshinone IIA; Tetrandrine; Carvacrol; cis-6-Nonenol; Retinyl punicate;Retinyl oleate; Equol; MycoFusions Coriolus Black Corn Biomass;MycoFusions Maitake Waxy Hulless Barley Biomass; Zanthoxylum nitidiumextract; Ophiopogon Thunb. P.E. extract; Radix platycodonis extract;Terminalia belerica extract; Cocculus glaucescens extract; Stephaniasolid extract; and Rosemary extract. In some embodiments, the cosmeticcomposition comprises one or more paxillin stimulators, and in otherembodiments, the cosmetic composition comprises two or more paxillinstimulators.

In some embodiments, the cosmetic composition comprises cis-6-nonenol asthe paxillin stimulator in an amount effective to improve the aestheticappearance of skin, such as to impart an anti-aging benefit to the skin.In another embodiment, the cosmetic composition comprises cis-6-nonenolas a first paxillin stimulator and at least one other paxillinstimulator, the cis-6-nonenol and the other paxillin stimulator beingpresent in the composition in an aggregate amount effective to impart ananti-aging benefit to the skin

In some embodiments, one of more of the substances of the group areexcluded from the cosmetic composition. For example, in someembodiments, the composition does not include Trifolium hybridum extractand/or does not include retinyl oleate and/or does not include Equol.

In some embodiments, the cosmetic composition further comprises at leastone other skin active, said skin active being selected from the groupconsisting of botanicals, keratolytic agents, desquamating agents,keratinocyte proliferation enhancers, elastase inhibitors, depigmentingagents, anti-inflammatory agents, steroids, anti-acne agents,antioxidants, salicylic acid or salicylates, thiodipropionic acid oresters thereof, advanced glycation end-product (AGE) inhibitors, andalpha-hydroxyacids. In some embodiments, the cosmetic compositionfurther comprises a collagen stimulator, such as TDPA.

In another aspect, the invention relates to methods comprising topicallyapplying to the skin a cosmetic composition comprising one or morepaxillin stimulators. The cosmetic composition will comprise aneffective amount of at least one paxillin stimulator to provide ananti-aging benefit to human skin, such as, to treat, reverse, ameliorateand/or prevent signs of skin aging. Anti-aging benefits include withoutlimitation, the following:

(a) treatment, reduction, and/or prevention of fine lines or wrinkles,

(b) reduction of skin pore size,

(c) improvement in skin thickness, plumpness, and/or tautness;

(d) improvement in skin suppleness and/or softness;

(e) improvement in skin tone, radiance, and/or clarity;

(f) improvement in procollagen and/or collagen production;

(g) improvement in skin texture and/or promotion of re-texturization;

(h) improvement in skin barrier repair and/or function;

(i) treatment and/or prevention of skin sagging or atrophy; and/or

(j) improvement in appearance of skin contours;

(k) restoration of skin luster and/or brightness;

(l) replenishment of essential nutrients and/or constituents in theskin;

(m) improvement of skin appearance decreased by menopause;

(n) improvement in skin moisturization and/or hydration; and

(o) improvement of skin elasticity and/or resiliency.

Also provided is a method for imparting an anti-aging benefit to humanskin comprising topically applying to skin in need thereof a compositioncomprising at least one paxillin stimulator in an amount effective toincrease paxillin level in dermal fibroblasts, e.g., by up-regulatingpaxillin expression. Skin in need thereof can photo-aged and/or skindamaged by UV radiation.

In another aspect of the invention, a method of treating, reversing,ameliorating and/or preventing fine lines or wrinkles or sagging inhuman skin is provided, comprising topically applying to skin in needthereof, including applying directly to a wrinkle or fine line, acomposition comprising at least one paxillin stimulator in an amounteffective to increase the level of paxillin in dermal fibroblasts.

In still another aspect, the invention relates to methods andcompositions for assaying whether a candidate material is a paxillinstimulator. Screening methods can comprise contacting a human skin cellwith a candidate material and assaying for up-regulation of paxillinmRNA. Assaying for up-regulation of paxillin mRNA may comprise abranched DNA technology using labeled probes specifically designed toidentify paxillin mRNA. In some embodiments, the probes comprisenucleotide sequences that together cover the paxillin mRNA sequence. Insome embodiments, for example, the probes comprise nucleotide sequencesaccording to SEQ ID NOS: 1, 2, and/or 3, disclosed herein, and/orvariations thereof. Still other aspects of the invention relate tocandidate materials identified as paxillin stimulators, e.g., candidatematerials identified as up-regulating paxillin mRNA using one or moreprobes, compositions, and/or methods described herein.

In still another aspect, the invention relates to methods forformulating a cosmetic composition for imparting an anti-aging benefitto human skin comprising assaying to determine if a candidate materialis a paxillin stimulator; and, if so, incorporating the material into acosmetically acceptable vehicle. In some embodiments, the candidatematerial is a plant extract.

These and other aspects of the present invention will be betterunderstood by reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates human skin biopsies showing paxillin protein levelsafter topical treatment with (A) a control vehicle; and (B) a candidatepaxillin stimulator.

DETAILED DESCRIPTION

It has surprisingly been found that compositions comprising one or moresubstances that stimulate paxillin can be topically applied to humanskin to improve the aesthetic appearance of the skin, in particular, totreat, reverse, and/or prevent visible signs of aging. Novel substancesas well as a number of substances previously known in the cosmetic artshave surprisingly been found to stimulate paxillin and hence will finduse in topical cosmetic compositions with anti-aging benefits.

In view of these findings and others, a topical composition comprisingone or more paxillin stimulators is contemplated to be useful incombating signs of skin aging, including reducing fine lines andwrinkles, preserving skin suppleness and softness, preventing skinsagging or atrophy, improving skin plumpness and/or tautness andcounteracting other signs of skin aging and/or skin damage. It isfurther contemplated that other substances that stimulate paxillinproduction or otherwise promote increased intracellular paxillin levelsin dermal fibroblasts can find use in anti-aging topical compositions,according to the instant disclosure. As used herein, “consistingessentially of” means that the composition or component may includeadditional ingredients, but only if the additional ingredients do notmaterially alter the basic and novel characteristics of the claimedcompositions or methods.

Paxillin Stimulating Compounds and Compositions

One aspect of the present invention relates to compositions for topicalapplication which comprise one or more paxillin stimulators to impart ananti-aging benefit to the skin, such as treating, reversing,ameliorating, delaying and/or preventing signs of skin aging. Combatingsigns of skin aging may include without limitation, the following:

(a) treatment, reduction, and/or prevention of fine lines or wrinkles,

(b) reduction of skin pore size,

(c) improvement in skin thickness, plumpness, and/or tautness;

(d) improvement in skin suppleness and/or softness;

(e) improvement in skin tone, radiance, and/or clarity;

(f) improvement in procollagen and/or collagen production;

(g) improvement in skin texture and/or promotion of retexturization;

(h) improvement in skin barrier repair and/or function;

(i) treatment and/or prevention of skin sagging or atrophy; and/or

(j) improvement in appearance of skin contours;

(k) restoration of skin luster and/or brightness;

(l) replenishment of essential nutrients and/or constituents in theskin;

(m) improvement of skin appearance decreased by menopause;

(n) improvement in skin moisturization and/or hydration; and

(o) improvement of skin elasticity and/or resiliency.

In practice, the compositions of the invention are applied to skin inneed of treatment, that is, skin which suffers from a deficiency or lossin any of the foregoing skin attributes or which would otherwise benefitfrom improvement in any of the foregoing skin attributes.

A “paxillin stimulator” refers to any agent that can increase the levelof paxillin in human dermal fibroblasts. Increase in paxillin levels canrefer to an increase in paxillin mRNA transcribed and/or an increase inpaxillin protein expressed in the fibroblast, in vitro or in vivo, andcan be measured by any means known in the art, or as described herein.For example, the paxillin stimulator can act as an up-regulator ofpaxillin expression within dermal fibroblasts. See, e.g., Example 1below. In some embodiments, paxillin level is increased by at leastabout 10%, at least about 20%, at least about 60%, at least about 80%,at least about 100%, at least about 150%, at least about 200%, at leastabout 250%, or at least about 300%, compared to the level of paxillin inthe absence of the paxillin stimulator. A “paxillin stimulator” may alsoinclude agents that bring about an effective increase in paxillin levelsby, e.g., increasing the stability of paxillin RNA and/or protein,and/or increasing localization of paxillin to sites of action.

The paxillin stimulator may be any organic or inorganic compound, smallmolecule, macromolecule, macromolecular complex, cellular lysate,sub-cellular compartment, extract of biological origin, or combinationthereof.

In some embodiments, the paxillin stimulator comprises an organicmolecule. In some embodiments, the paxillin stimulator comprises apyridone-fused azabicyclic compound, or a pharmaceutically orcosmetically acceptable salts and/or prodrug thereof. The pyridone-fusedazabicyclic compounds include, without limitation, thepaxillin-stimulating compounds according to formula (I):

wherein, R₁, R₂, R₃, and R₄ are independently selected from hydrogen;—R; halogen (—F, —Cl, —Br, —I); —OH; —C≡C—R; —C≡N; —C(R)═N—R^(N);—C═N—N(R^(N))₂; —C(═NR_(N))—N(R^(N))₂; —CH₂OH; —CHO; —(C═O)—R; —CO₂H;—CO₂ ⁻; —CO₂R; —CS₂R; —(C═O)—S—R; —S—(C═O)—R; —(C═O)—NH₂;—(C═O)—NR^(N)R^(N); —(C═O)—NHNH₂; —O—(C═O)—NHNH₂; —(C═S)—NH₂;—(C═S)—N(R^(N))₂; —O—(C═O)—H; —O—(C═O)—R; —O—(C═O)—NH₂;—O—(C═O)—NR^(N)R^(N); —OR; —SR; —NH₂; —NHR^(N); —NR^(N) ₂; —N(R^(N))₃;—N(R^(N))—OH; —N(→O)(R)₂; —O—N(R^(N))₂; —N(R^(N))—O—R;—N(R^(N))—N(R^(N))₂; —NR^(N)—(C═O)—R; —NR^(N)C(═O)O—R; —NR^(N)—CHO;—NR^(N)—(C═O)—R; —NR^(N)C(═O)NR^(N); —N(R^(N))—C(═O)—N(R^(N))₂;—N(R^(N))—C(═S)—N(R^(N))₂; —N═CR₂; —N═N—R^(N); —SCN; —NCS; —NSO; —SS—R;—SO—R; —SO₂—R; —O—S(═O)₂—R; —S(═O)₂—OR; —N(R^(N))—SO₂—R; —SO₂—N(R)₂;—O—SO₃ ⁻; —O—S(═O)₂—OR; —O—S(═O)—OR; —O—S(═O)—R; —S(═O)—OR; —S(═O)—R;—NO; —NO₂; —NO₃; —O—NO; —O—NO₂; —N₃; —N₂; —N(C₂H₄); —Si(R)₃; —CF₃;—O—CF₃; —(C═O)—R; —PR₂; —O—P(═O)(OR)₂; and —P(═O)(OR)₂; and where anytwo adjacent groups R₁, R₂ and R₃ may together form a five- orsix-membered ring fused to the pyridone ring;

L is either a bond (i.e., L is omitted), or is a moiety selected fromthe group consisting of —(C═O)—, —C(O)—P—, —(C═O)—NR^(N)—, —(CH₂)_(a)—,—(C═O)—(CH₂)_(a)—C(O)—, —(C═O)—(CH₂)_(a)—, —(CH₂)_(a)—C(O)—, where “a”is an integer from 1 to 6; and where L is preferably —(C═O)—;

and R and R^(N) are selected independently at each occurrence fromhydrogen or a C₁-C₃₀ hydrocarbon radical, optionally including from 1-20heteroatoms, such as oxygen, sulfur, and nitrogen atoms; where R andR^(N) are preferably selected from substituted or unsubstituted,branched, straight chain or cyclic, C₁-C₂₀ alkyl, alkenyl, alkynyl,aryl, benzyl, heteroaryl, alkyl-aryl, aryl-alkyl, alkyl-heteroaryl,heteroaryl-alkyl, heteroaryl-aryl, aryl-heteroaryl, bicyclic alkyl,aryl, or heteroaryl radicals, and combinations thereof; and wherein eachof the foregoing radicals may be substituted with 1-6 heteroatoms and/orwith any heteroatom-containing moiety, including, for example, acyl,acyloxy, amino, alkoxyl, alkylamino, alkylthiol, alkylimino,alkylsulfonate, amide, azo, carboxyl, carboxamide, carbamide, cyano,dialkylamino, ester, halogen, hydroxyl, nitro, oxo, oxa, oxime,perfluoro, phosphate, phosphonyl, phosphinyl, sulfate, sulfate,sulfo-alkyl, thiol, thioether, thioester, thioalkoxy, thiocyanate, andcombinations thereof.

In some embodiments according to formula (I), R₁ is a group —R, where Ris preferably selected from substituted or unsubstituted, branched,straight chain or cyclic, C₁-C₂₀ alkyl, alkenyl, alkynyl, aryl, benzyl,heteroaryl, alkyl-aryl, aryl-alkyl, alkyl-heteroaryl, heteroaryl-alkyl,heteroaryl-aryl, aryl-heteroaryl, bicyclic alkyl, aryl, or heteroarylradicals, and combinations thereof; and wherein each of the foregoingradicals may be substituted with 1-6 heteroatoms and/or with anyheteroatom-containing moiety, including, for example, acyl, acyloxy,amino, alkoxyl, alkylamino, alkylthiol, alkylimino, alkylsulfonate,amide, azo, carboxyl, carboxamide, carbamide, cyano, dialkylamino,ester, halogen, hydroxyl, nitro, oxo, oxa, oxime, perfluoro, phosphate,phosphonyl, phosphinyl, sulfate, sulfo-alkyl, thiol, thioether, thia,thioalkoxy, thiocyanate, and combinations thereof.

R may comprise, for example, aliphatic C₁-C₂₀ hydrocarbon radicals;including aliphatic C₁-C₁₂ hydrocarbon radicals, aliphatic C₁-C₈hydrocarbon radicals, or aliphatic C₁-C₆ hydrocarbon radicals, asexemplified by substituted or unsubstituted branched, straight chain orcyclic, alkyl, alkenyl (e.g., vinyl, allyl, etc.), and alkynyl moieties;C₆-C₂₀ aromatic hydrocarbon radicals, including C₆-C₁₂ aromatichydrocarbon radicals, C₆-C₁₀ aromatic hydrocarbon radicals, or C₆aromatic hydrocarbon radicals, as exemplified by substituted orunsubstituted aryl (e.g., phenyl), alkyl-aryl (e.g., benzyl), aryl-alkyl(e.g., toluoyl), and the like; or C₁-C₂₀ heteroaryl radicals includingone or more heteroatoms selected from O, N, and S in the ring; includingC₁-C₁₂ heteroaromatic radicals, C₁-C₈ heteroaromatic radicals, and C₁-C₆heteroaromatic radicals, as exemplified by heteroaryl, alkyl-heteroaryl,heteroaryl-alkyl and the like. In some embodiments, R may be, forexample, independently at each occurrence, hydrogen, methyl, ethyl,propyl, butyl, pentyl, hexyl, phenyl, benzyl, or the like, includinghalo and perhalo derivatives thereof.

In some embodiments, R₂ and/or R₃ will be hydrogen. In some embodiments,L is a group —(C═O)—. In other embodiments according to formula (I), R₁is a group —R, where R is an aryl or heteroaryl group, either of whichmay be optionally substituted with a group —R and/or with 1-6heteroatoms and/or with any of the heteroatom-containing moietiesdescribed above. Typically, where R is an aryl or heteroaryl group, thering will comprise five or six atoms in the ring system. In particular,paxillin-stimulating compounds according to formula (II) arecontemplated to be useful:

where R₁ is as defined above, or is a group -Q(-R*)_(n), where Q is ringhaving three, four, five or six atoms in the ring, including aromatic orheteroaromatic rings, and R* represents substitution on the ring and isas defined above for R and is independently selected at each occurrence.The substituents on ring Q, represented by R*, may be attached to anyavailable ring atom, except for the point of attachment to the pyridonein the case of aromatic and heteroaromatic rings Q. The number ofsubstituents is determined by “n” which is an integer from 0 (i.e., R*is absent) to 5, depending on the number of positions available forsubstitution on the ring; and where R₂, R₃ and R₄ are as defined above.In some embodiments according to formula (II), R₂ and/or R₃ arehydrogen.

Non-limiting examples of three-membered heterocyclic rings, include butare not limited to, aziridine, oxirane, thiirane, diaziridine, andoxaziridine. Non-limiting examples of four-membered heterocyclic rings,include but are not limited to, azetidine, oxetane, thietane,diazetidine, oxazetidine, and 1,2-oxathietane.

Five membered heterocycles represent a currently preferred embodiment ofthe invention for the substituent Q. Non-limiting examples offive-membered heterocylic rings include, without limitation,pyrrolidine, tetrahydrofuran, tetrahydrothiophene, oxazolidine,thiazolidine, 1,3-dioiane, 1,3-oxzthiolane, 1,3-dithiolane,imidazolidine, pyrazolidine, pyrrole, furan, thiophene, oxazole,isoxazole, thiazole, isothiazole, imidazole, pyrazole, 1,3,4-oxadiazole,1,2,4-oxadiazole, 1,3,4-thiadiazole, 1,2,4-thiadiazole, 1,3,4-triazole,1,2,3-triazole, and the like. Q may be selected from, for example, thefollowing five membered heterocyclic rings which are aromatic, fullysaturated, or comprises one, or two double bonds:

These five membered rings may be optionally functionalized with one ormore groups R*, as defined above, with particular mention being made ofC₁₋₄ alkyl (e.g., methyl, ethyl, isopropyl, etc.), C₁₋₄ alkoxyl, halo,hydroxyl, oxo, thiol, amino, alkylamino, dialkylamino, —CH₂—N(CH₃)₂,—(CH₂)₁₋₆—N(R^(N))₂, —(C═O)—H, —(C═O)—R, —(C═O)—CH₃, —(C═O)—CH₂CH₃,—O—(C═O)—R, —O—(C═O)—CH₃, and —(C═O)—O—R. Further, any nitrogen atom maybe optionally oxidized to the N-oxide, and any sulfur atom may beoptionally oxidized to a sulfoxide.

Non-limiting examples of six-membered rings which are suitably selectedfor Q include, without limitation, 2H-pyran, tetrahydropyran,piperidine, 1,4-dioxane, morpholine, piperazine, 1,4-dithiane,thiomorpholine, pyridine, pyrazine, pyridazine, pyrimidine,1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, 1,2,3,4-tetrazine, andpentazine, to name a few. Q may be selected from, for example, thefollowing six membered heterocyclic rings which are aromatic, fullysaturated, or comprises one, two, or three double bonds:

These six membered rings may be optionally functionalized with one ormore groups R*, as defined above, with particular mention being made ofC₁₋₄ alkyl (e.g., methyl, ethyl, isopropyl, etc.), C₁₋₄ alkoxyl, halo,hydroxyl, oxo, thiol, amino, alkylamino, dialkylamino, —CH₂—N(CH₃)₂,—(CH₂)₁₋₆—N(R^(N))₂, —(C═O)—H, —(C═O)—R, —(C═O)—CH₃, —(C═O)—CH₂CH₃,—O—(C═O)—R, —O—(C═O)—CH₃, and —(C═O)—O—R. Further, any nitrogen atom maybe optionally oxidized to the N-oxide, and any sulfur atom may beoptionally oxidized to a sulfoxide.

In one embodiment, Q is phenyl and “n” is 1, 2, or 3. In one interestingembodiment, Q is phenyl, “n” is 1, and R* is a group—(CH₂)₁₋₆—N(R^(N))₂, and in particular —CH₂—N(CH₃)₂. In this embodiment,R* may by para, meta, or ortho to the point of attachment to thepyridone ring, as illustrated in formula (III).

In this and all embodiments, R* and/or R₄ may be a lower (C₁-C₆)aliphatic group, including for example, methyl, ethyl, n-propyl,isopropyl, cyclopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,cyclobutyl, 1-methylbutyl, 1,1-dimethylpropyl, n-pentyl, cyclopentyl,iso-pentyl, neo-pentyl, n-hexyl, and cyclohexyl, or the like. Theselower alkyl group may also include 1-6 heteroatoms selected from O, S,and N in the chain or attached to the chain. Representative groups forR* and/or R₄ include, but are not limited to, —(CH₂)_(a)—CH₃ where “a”is an integer from 1 to 5, including, for example, —CH₂—CH₃, —CH₂CH₂CH₃,—CH₂CH₂CH₂CH₃, or —CH₂CH₂CH₂CH₂CH₃; linear alkoxy moieties of thegeneral form —O(CH₂)_(a)—CH₃ where “a” is an integer from 1 to 5,including for example, —OCH₂CH₃, —OCH₂CH₂CH₃, or —OCH₂CH₂CH₂CH₃; groupsof the form —O—(CH₂)_(a)—O—(CH₂)_(b)CH₃ where “a” and “b” are eachindependently integers from 1 to 4; —(CH₂)_(a)—O—(CH₂)_(b)CH₃ or—(CH₂)_(a)S(CH₂)_(b)CH₃, where “a” and “b” are each independentlyintegers from 1 to 4; or a moiety of the form —(CH₂)_(b)NR^(N) ₂ whereR^(N) is independently at each occurrence a group —(CH₂)_(c)CH₃ where“b” is an integer from 1 to 4 and “c” is an integer from 0 (zero) to 4.Specific examples of R* and/or R₄ include, without limitation, —O—CH₃,—O—CH₂CH₃, —O—CH₂CH₂CH₃, —CH₂—O—CH₃, —CH₂—O—CH₂CH₃, —CH₂CH₂—O—CH₃,—N(CH₃)₂, —N(CH₂CH₃)₂, —N(CH₂CH₂CH₃)₂, —S—CH₃, —S—CH₂CH₃, —CH₂—S—CH₃,—CH₂—S—CH₂CH₃, —CH₂CH₂—S—CH₃, —S—CH₂CH₂CH₃, —CH₂CH₂—S—CH₃,—CH₂—S—CH₂CH₃, —CH₂—N(CH₃)₂, —CH₂—N(CH₂CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂,—CH₂—N(CH₂CH₂CH₃)₂, —CH₂CH₂—N(CH₃)₂, and —CH₂—N(CH₂CH₃)₂, to name a few.

In one embodiment, R* is a group —(CH₂)₁₋₆—N(R^(N))₂, and in particular—CH₂—N(CH₃)₂. In another embodiment, R₄ is a group —CH₂—S—CH₃. Excellentresults have been obtained with the compounds according to formulas(IV-A) and (IV-B), shown below, where R* comprises a tertiary aminegroup ortho or para to the point of attachment to the pyridone ring andR₄ comprises a linear thioether moiety:

In formulas (I)—(III), R₄ may, for example, also be a group a group-Q(-R)—, where Q is, independent from R₁, any of the cyclic groupsdefined above. In some embodiments, R₁ and R₄ are, independently, agroup -Q(-R)—, where Q is the same or different at both occurrences. Inone embodiment, R₁ is an optionally substituted five- or six-memberedaromatic group, such as the five-membered furyl group, and R₄ is anoptionally substituted five- or six-membered aromatic group, such asphenyl group. The compound according to formula (V), has been founduseful:

In addition to the paxillin-stimulating compounds according to formulae(I)—(V), other pyridone-fused azabicyclic compounds capable ofstimulating paxillin are contemplated, including without limitation,those described in International Patent Application Publication WO98/18798; EP 0 581 457; U.S. Pat. Nos.6,630,467, 6,235,734, thedisclosures of which are incorporated herein by reference in theirentirety.

In some embodiments, the paxillin stimulator comprises another kind oforganic molecule, such as, e.g., Tanshinone IIA; Tetrandrine; Carvacol;and/or cis-6-Nonenol, and/or salt thereof. Tanshinone IIA, or1,6,6-trimethyl-8,9-dihydro-7H-naphtho[1,2-g][1]benzofuran-10,11-dione,is a diterpenoid naphthoquinone and an active component of sometraditional Chinese medicines based on Salvia miltiorrhiza. It isbelieved to have anti-inflammatory activity and induces apoptosis in avariety of cell lines. H. J. Sung et al. Exp. Mol. Med. 1999 Vol.31:174. It has also been suggested for use in treating sepsis,septicemia, and endotoxic shock. See, e.g., International PatentApplication Publication WO 2007/084419, which is incorporated herein byreference in its entirety. Tanshinone IIA is commercially available,e.g., it can be obtained from Biomol Research Laboratories, PA.

Tetrandrine, or 6,6′,7,12-Tetramethoxy-2,2′-dimethyl-berbaman, is abis-coclaurine alkaloid, which has been used for centuries in Chinesetraditional medicines, for example, for treating cardiovasculardiseases. It is believed to be a calcium channel blocker as well as ablocker of the Ca²⁺-activated potassium channels. Dworetzky et al. J.Neurosci. 1996. Vol. 16: 4543; and Gadwood et al. Annu Rep. Med. Chem.1989 Vol. 24: 187. It is also commercially available, e.g., from BiomolResearch Laboratories, PA.

Carvacrol, or cymophenol, C₆H₃CH₃(OH)(C₃H₇), is a monoterpenoid phenol,having a characteristic pungent, warm odor, similar to that of oregano,and is also known if its pizza-like taste. Ultee A. et al. (2000) J.Food Prot. 63 (5): 620-4. Carvacrol is present in oils of Origanumvulgare, thyme, pepperwort, and wild bergamot. It is known to inhibitgrowth of a number of bacteria strains and accordingly has been used asa food additive to prevent bacterial contamination. Ultee A. et al.(2001) Int. J. Food Microbiol. 64 (3): 373-8.

Cis-6-Nonenol is an unsaturated fatty alcohol. Cis-6-nonenol has thestructure of formula (VI), shown below, and has been further describedin U.S. patent application Ser. No. 12/342,197, filed Dec. 23, 2008,entitled “Topical Compositions Containing cis-6-Nonenol and itsDerivatives and Methods for Treating Skin” incorporated herein byreference in its entirety.

In some embodiments, the paxillin stimulator comprises a larger organicmolecule, such as, e.g., retinyl punicate; retinyl oleate; and/or Equol.Retinyl punicate and retinyl oleate are esters of retinol (vitamin A)with the fatty acids punicic acid and oleic acid, respectively. Theesters can be obtained as known in the art, e.g., by esterificationreaction. Retinyl oleate has been suggested for use in enhancing skinand for treating dermatological disorders, including acne, oily skin,and rosacea. See, e.g., EP 0 807 429, which is incorporated herein byreference in its entirety Equol, or 4′,7-isoflavandiol, has beensuggested for use in treating androgen-mediated pathologies of skin andhair, by blocking the hormonal action of 5-dihydrotestosterone. See,e.g., International Patent Application Publication WO 2005/107770, whichis incorporated herein by reference in its entirety. It is anisoflavandiol and nonsteroidal estrogen, made by bacterial flora in theintestines of some individuals. Wang, X. L., et al. Applied andEnvironmental Microbiology (2005) 71:214-219; Frankenfeld, C. L. et al.The British journal of Nutrition (2005) 94:873-876.

In some embodiments, the paxillin stimulator comprises a biologicalextract, e.g., a plant extract. Extracts of plants useful as paxillinstimulators in the present invention include without limitation Jasminumsambac extract; Coccinia grandis extract; Ecliptica prostrata Linn.extract; Clitoria ternatea Linn. extract; Ozothamnus obcordatus extract;Erythrina flabelliformis extract; Lonchocarpus capassa extract; Sophoratomentosa extract; Trifolium hybridum extract; Eremophila mitchelliiextract; Kunzea ambigua extract; Zanthoxylum nitidium extract;Ophiopogon Thunb. P.E. extract; Radix platycodonis extract; Terminaliabelerica extract; Cocculus glaucescens extract; Stephania solid extract;and/or rosemary extract, preferably rosemary PE 50%.

Jasminum sambac is a species of jasmine native to southwestern andsouthern Asia, the Philippines, India, Myanmar and Sri Lanka. Itproduces strongly-scented flowers, used as ornaments, to make fragrantleis, and as the main ingredient in jasmine tea. Coccinia grandis, alsoknown as ivy gourd, is a tropical vine grown in some parts of the worldfor its small edible fruits. The fruit is commonly eaten, e.g., inIndian cuisine, whereas the vine is considered an invasive plant in theUnited States and Hawai'i. Coccinia grandis has been suggested as usefulin skin whitening applications, e.g., see JP 2000095663, which isincorporated herein by reference in its entirety. Ecliptica prostrataLinn., also known as False Daisy or bhringraj, grows as a week in moistplaces, including India, China, Brazil, and Thailand, and is used inmany traditional medicines in those areas. For example, in Brazil it isused to treat snakebites and in India is it used to improve hair growth.Chopra, et al. 1955. Glossary of Indian Medicinal plants. C.S.I.R., NewDelhi. Chinese traditional medicine applications for the plant includetreating athlete's foot, eczema, and dermatitis. Clitoria ternatea, alsoknown as Butterfly pea, is native to tropical and temperate areas,including southeast Asia. The flowers can be used as a food dye andextracts of the roots are considered in some cultures to have medicinalproperties, such as treating whooping cough. Ozothamnus obcordatus, alsoknown as Grey Everlasting, is a shrub native to some parts of Australiaand is regarded as having commercial cut-flower potential.

Erythrina flabelliformis, also known as Coral Bean, is shrub bearing redflowers in showy clusters, as well as large pods with highly toxic redbeans. It has been suggested for use in extractions to obtain usefulchemicals from plants. See, e.g., U.S. Pat. Appl. Publ. No.2002/0132021, which is incorporated herein by reference in its entirety.Lonchocarpus capassa, also known as apple-leaf or rain tree, is found insouthern Africa. It is known for its smooth bare trunk with ahigh-branching sparse canopy. The tree is often infected with aphids,which excrete drippings to form wet patches beneath the tree, giving itthe name “rain tree.” Sophora tomentosa, also known as “necklace pod”,is a shrub bearing yellow flowers and silver, velvety foliage. The plantis native to coastal areas of Florida and the Caribbean. Trifoliumhybridum, also known as “alsike clover”, is a plant in the pea family,bearing stalked, pale pink flowers, sometimes grown as fodder. Plantextracts have been used in dermatological applications, ostensibly asinhibitors of extracellular proteases. See, e.g., U.S. Pat. Appl. Publ.No. 2007/0122492, which is incorporated herein by reference in itsentirety. Eremophila mitchellii, also known as “false sandalwood”, is asshrub or small tree native to Australia that bears white or pale pinkishflowers. Kunzea ambigua, also known as “poverty bush”, is a shrub foundgrowing in sandstone soils of eastern Australia. It is used in gardeningand sand dune stabilization. It has also been suggested for use intreating psoriasis. See, e.g., International Patent ApplicationPublication WO 2009/086595, which is incorporated herein by reference inits entirety.

Zanthoxylum nitidium is a shrub known for the use of its roots andsometimes bark in traditional Chinese medicine. Several alkaloidsthought to contribute to its medicinal qualities have been isolated fromextracts of this plant. Mingjin L. et al. (2006) J. of Pharm. andBiomed. Anal. 42(2):178-183. Ophiopogon Thunb. is a grass-like shrubwidely cultivated in China for its tuberous roots, which are usedmedicinally. Extracts can by obtained commercially, e.g., from WuchangYuancheng Technology Development Co., Ltd., China. Radix platycodonis isanother plant used in traditional Chinese medicine. For example, itsroots can be dried to form a brown powder that is availablecommercially, e.g., from Qi Lu Chemical, China. Terminalia belerica,also known as bibhitaka, is a tall tree growing throughout India withbrownish grey bark and offensive-smelling flowers. In Sanskrit,babhitaka means something that keeps away disease, and parts of the treeare used extensively in traditional medicines. Extracts also have beensuggested for use as de-pigmenting agents. See, e.g., InternationalPatent Application Publication WO 96/24327, which is herein incorporatedby reference in its entirety. Cocculus glaucescens is a large, woodyvine used in traditional Chinese medicine. Stephania is a genus offlowering plants that grows as perennial vines native to eastern andsouthern Asia and Australia. Some species provide herbs used intraditional Chinese medicine and solid extracts of the plant parts,e.g., the roots are commercially available, e.g., from NaturalNutritionals, GA, as well as from Naturex, Inc, NJ, which provides anStephania solid extract suitable for use in accordance with theinvention. Rosemary, Rosmarinus officinalis, is a woody perennial herbwith fragrant evergreen needle-like leaves, native to the Mediterraneanregion and extensively used in traditional Mediterranean cuisine.Rosemary extract suitable for use in the invention is commerciallyavailable, e.g., from Naturex, Inc., NJ.

In some embodiments, the paxillin stimulator comprises other biologicalmaterials, e.g., MycoFusions Coriolus Black Corn Biomass and/orMycoFusions Maitake Waxy Hulless Barley Biomass. MycoFusions CoriolusBlack Corn Biomass and MycoFusions Maitake Waxy Hulless Barley Biomassare commercially available, e.g., from Nutragenesis, LLC, VT.

The cosmetic composition for topical application to the skin willcomprise at least one paxillin stimulator in a cosmetically acceptablevehicle. The paxillin stimulator may be at least one substance selectedfrom the group consisting of pyridone-fused azabicyclic compounds havingthe structure of formulae IV or V; Jasminum sambac extract; Cocciniagrandis extract; Eliptica prostrata Linn. extract; Clitoria ternateaLinn. extract; Ozothamnus obcordatus extract; Erythrina flabelliformisextract; Lonchocarpus capassa extract; Sophora tomentosa extract;Trifolium hybridum extract; Eremophila mitchellii extract; Kunzeaambigua extract; Tanshinone IIA; Tetrandrine; Carvacrol; cis-6-Nonenol;Retinyl punicate; Retinyl oleate; Equol; MycoFusions Coriolus Black CornBiomass; MycoFusions Maitake Waxy Hulless Barley Biomass; Zanthoxylumnitidium extract; Ophiopogon Thunb. P.E. extract; Radix platycodonisextract; Terminalia belerica extract; Cocculus glaucescens extract;Stephania solid extract; and Rosemary PE 50%. In some embodiments, thecosmetic composition comprises one or more paxillin stimulators, and inother embodiments, the cosmetic composition comprises two or morepaxillin stimulators. In preferred embodiments, the combination of twoor more paxillin stimulators produces synergistic effects, such assynergistically providing anti-aging benefits.

In some embodiments, the cosmetic composition for topical application tothe skin comprises, in a cosmetically acceptable vehicle, cis-6-nonenoland at least one other paxillin stimulator in amounts effective toimpart an anti-aging benefit to human skin. The paxillin stimulator maybe at least one compound selected from the group consisting ofpyridone-fused azabicyclic compounds having the structure of formulae IVor V; Jasminum sambac extract; Coccinia grandis extract; Elipticaprostrata Linn. extract; Clitoria ternatea Linn. extract; Ozothamnusobcordatus extract; Erythrina flabelliformis extract; Lonchocarpuscapassa extract; Sophora tomentosa extract; Trifolium hybridum extract;Eremophila mitchellii extract; Kunzea ambigua extract; Tanshinone IIA;Tetrandrine; Carvacrol; Retinyl punicate; Retinyl oleate; Equol;MycoFusions Coriolus Black Corn Biomass; MycoFusions Maitake WaxyHulless Barley Biomass; Zanthoxylum nitidium extract; Ophiopogon Thunb.P.E. extract; Radix platycodonis extract; Terminalia belerica extract;Cocculus glaucescens extract; Stephania solid extract; and Rosemary PE50%. In some embodiments, the composition comprising cis-6-nonenolfurther comprises at least two, at least five, at least 8, at least 10,at least 15, at least 20, at least 25, or all 28 substances selectedfrom the group recited above. In preferred embodiments, the combinationof two or more paxillin stimulators produces synergistic effects, suchas synergistically providing anti-aging benefits.

In some preferred embodiments, the cosmetic composition comprises atleast one paxillin stimulator selected from the group consisting ofpyridone-fused azabicyclic compounds having the structure of formulae IVor V; Jasminum sambac extract; Eliptica prostrata Linn. extract;Clitoria ternatea Linn. extract; Ozothamnus obcordatus extract;Erythrina flabelliformis extract; Lonchocarpus capassa extract; Sophoratomentosa extract; Tetrandrine; Carvacrol; Retinyl punicate; MycoFusionsCoriolus Black Corn Biomass; MycoFusions Maitake Waxy Hulless BarleyBiomass; Zanthoxylum nitidium extract; Ophiopogon Thunb. P.E. extract;Radix platycodonis extract; and Cocculus glaucescens extract. In somepreferred embodiments, the paxillin stimulator comprises one or more ofTrifolium hybridum extract, Kunzea ambigua extract, Tanshinone IIA,Equol, Terminalia belerica extract, or Rosemary extract. In someembodiments, one of more of Trifolium hybridum extract, Kunzea ambiguaextract, Tanshinone HA, Equol, Terminalia belerica extract, and Rosemaryextract is used in combination with cis-6-nonenol to impart anti-agingbenefit to skin.

In some preferred embodiments, one of more of the substances areexcluded from the cosmetic composition. For example, in someembodiments, the composition does not include Trifolium hybridum extractand/or does not include retinyl oleate and/or does not include Equol.

Cosmetic compositions of the instant invention generally comprise anamount of a paxillin stimulator, e.g., an amount of cis-6-nonenol,effective to provide a benefit to human skin. In preferred embodiments,the compositions comprise an amount of a paxillin stimulator effectiveto increase paxillin transcription and/or expression in human dermalfibroblasts. Cosmetic compositions described herein find use asanti-aging agents, e.g., as detailed below.

Cosmetic Use of Paxillin Stimulating Compositions

Another aspect of the instant invention relates to cosmetic use ofcompositions comprising a paxillin stimulator, such as cis-6-Nonenol.The cosmetic compositions surprisingly act to increase paxillin levelsin human dermal fibroblasts and accordingly find use in anti-agingproducts.

In some embodiments, a method for providing at least one benefit tohuman skin is provided, where the method comprises topically applying toskin in need thereof at least one paxillin stimulator in a cosmeticallyacceptable vehicle. The composition will comprise an effective amount ofthe substance. An “amount effective” or an “effective amount” to providea particular benefit to the skin refers to the active amount of apaxillin stimulator sufficient to provide a clinically measurableimprovement in the particular manifestation of skin when applied for asufficient time. “Amounts effective” or “effective amounts” to provide aparticular benefit to the skin refers to the active amounts of each oftwo or more paxillin stimulators used in combination to provide aclinically measurable improvement in the particular manifestation ofskin when applied for a sufficient time. The effective amount of eachsubstance when used in combination with another may be the same, greaterthan, or less than the effective amount of the substance when usedalone. Use of lower amounts of individual substances is contemplatedwhen used in combination with other paxillin stimulators, e.g., due tosynergistic effects in producing a clinically measurable improvement ina particular manifestation of skin. Such benefits include withoutlimitation, the following:

(a) treatment, reduction, and/or prevention of fine lines or wrinkles,

(b) reduction of skin pore size,

(c) improvement in skin thickness, plumpness, and/or tautness;

(d) improvement in skin suppleness and/or softness;

(e) improvement in skin tone, radiance, and/or clarity;

(f) improvement in procollagen and/or collagen production;

(g) improvement in skin texture and/or promotion of retexturization;

(h) improvement in skin barrier repair and/or function;

(i) treatment and/or prevention of skin sagging or atrophy; and/or

(j) improvement in appearance of skin contours;

(k) restoration of skin luster and/or brightness;

(l) replenishment of essential nutrients and/or constituents in theskin;

(m) improvement of skin appearance decreased by menopause;

(n) improvement in skin moisturization and/or hydration; and

(o) improvement of skin elasticity and/or resiliency.

The compositions of the invention can be applied to skin in need oftreatment, such as skin which suffers from a deficiency or loss in anyof the foregoing attributes or conditions, or which would otherwisebenefit from the composition's anti-aging effects, e.g., as describedherein. For example, the paxillin stimulator(s) can be provided in acosmetically acceptable vehicle, topically applied to a desired area ofskin, and allowed to remain on the area in amount effective(s) to treatand/or prevent an unwanted feature or condition of the skin, and/or toimprove the aesthetic appearance of the skin.

“Condition of the skin” or “skin condition” is used interchangeablyherein with “skin disorder.” “Treatment” as used herein, as well asrelated terms such as “treat” or “treating,” refers to eradicating,reducing, ameliorating, or reversing one or more of the unwantedfeatures associated with the skin condition being treated, such that theconsumer perceives an improvement or other treatment benefit withrespect to the condition. “Prevention” as used herein, as well asrelated terms such as “prevent” or “preventing,” refers to affordingskin not yet affected by the condition a benefit that serves to avoid,delay, forestall, or minimize one or more unwanted features associatedwith the skin condition to be prevented. Such preventative benefitsinclude, for example, delaying development of the condition, or reducingthe duration, severity, or intensity of one or more unwanted featuresassociated with the condition if it eventually develops.

Anti-Aging Benefits

In certain preferred embodiments, the cosmetic compositions describedherein can be used to treat and/or prevent signs of skin aging or otherskin damage (e.g., from UV). Signs of skin aging include anydermatological signs of aging, including signs caused by intrinsic(chronological) aging, or caused by extrinsic factors (such as inphotoaging). The compositions may be applied to skin already showingvisible signs of aging, or likely to show such signs, e.g., due to ageor sun exposure.

An early sign of skin aging involves the gradual development of facialwrinkles, whether fine surface lines or deeper creases and folds. Whilewrinkling and other signs of aging are intrinsic to skin, the processmay be accelerated by external factors, such as excessive exposure tothe sun and other damaging elements, overactive facial expressionmuscles, frequent use of tobacco products, poor nutrition, or certainskin disorders. Fine surface lines that progress to deeper creases,deepening facial wrinkles due to repeated skin folding, and deep foldsthat develop with maturity are visible changes associated with aging.

Treating signs of skin aging refers to eradicating, reducing,ameliorating, or reversing one or more of the unwanted featuresassociated with skin aging, e.g., by reducing lines and/or wrinkles aperceptible extent. For example, compositions and methods of the instantinvention may be used to reverse or treat signs of skin aging oncemanifested, such as is common in individuals over 25 years of age.Preventing signs of skin aging refers to affording skin a benefit thatserves to avoid, delay, forestall, or minimize one or more unwantedfeatures associated with aging, e.g., by slowing the appearance of linesand/or wrinkles as the skin eventually ages. That is, the compositionsand methods of the instant invention may be employed prophylactically,e.g., to forestall signs of skin aging in individuals that have not yetmanifested signs of skin aging, most commonly in individuals under 25years of age.

The improvement in the unwanted feature and/or overall aestheticappearance can include one or more of the following: reducingdermatological signs of chronological aging, photo-aging, hormonalaging, and/or actinic aging; preventing and/or reducing the appearanceof lines and/or wrinkles; reducing the noticeability of facial lines andwrinkles, facial wrinkles on the cheeks, forehead, perpendicularwrinkles between the eyes, horizontal wrinkles above the eyes, andaround the mouth, marionette lines, and particularly deep wrinkles orcreases; preventing, reducing, and/or diminishing the appearance and/ordepth of lines and/or wrinkles; improving the appearance of suborbitallines and/or periorbital lines; reducing the appearance of crow's feet;rejuvenating and/or revitalizing skin, particularly aging skin; reducingskin fragility; preventing skin atrophy; improving skin tone, radiance,and/or clarity; preventing, reducing, and/or ameliorating skin sagging;improving skin firmness, plumpness, tautness, suppleness and/orsoftness; improving skin texture and/or promoting retexturization;improving skin barrier repair and/or function; improving the appearanceof skin contours; restoring skin luster and/or brightness; minimizingdermatological signs of fatigue and/or stress; resisting environmentalstress; replenishing ingredients in the skin decreased by aging and/ormenopause, such as essential nutrients or other skin constituents;ameliorating the effects of estrogen imbalance; improving communicationamong skin cells; increasing cell proliferation and/or multiplication;increasing skin cell metabolism decreased by aging and/or menopause;retarding cellular aging; improving skin moisturization and/orhydration; improving production and/or reducing loss of collagen and/orpro-collagen; enhancing skin thickness; increasing skin elasticityand/or resiliency; and any combinations thereof. Without wishing to bebound by theory, it is believed that the compositions of the presentinvention enhance and improve the aesthetic appearance of skin byenhancing paxillin expression and increasing paxillin levels in dermalfibroblasts.

In certain preferred embodiments, the compositions and methods of theinvention are directed to the treatment and/or prevention of fine linesor wrinkles in the skin. In the case of treatment, the compositions areapplied to skin in need of such treatment, by which is meant skin havingwrinkles and/or fine lines. The fine lines and/or wrinkles may occur onany surface of the skin, including without limitation, the skin of thehands, arms, legs, neck, chest, and face, including the forehead.Preferably, the compositions are applied directly to the fine linesand/or wrinkles For example, methods for treating fine lines andwrinkles may comprise topically applying a composition described hereinto skin of an individual in need thereof, e.g., topically applyingdirectly to a fine line and/or wrinkle in an amount and for a timesufficient to reduce the severity of the fine lines and/or wrinkles; orto inhibit formation of new lines and/or wrinkles The effect of acomposition on the appearance of fine lines and wrinkles can beevaluated qualitatively, e.g., by visual inspection, or quantitatively,e.g., by microscopic or computer assisted measurements of wrinklemorphology (e.g., the number, depth, length, area, volume and/or widthof wrinkles per unit area of skin).

The term “wrinkle” or “wrinkling” refers to both fine wrinkling and/orcoarse wrinkling Fine wrinkling or fine lines refers to superficiallines and wrinkles on the skin surface. Coarse wrinkling refers to deepfurrows, particularly deep lines/wrinkles on the face and around theeyes, including expression lines such as frown lines and wrinkles,forehead lines and wrinkles, crow's feet lines and wrinkles, nasolabialfolds, and marionette lines and wrinkles Forehead lines and wrinklesrefer to superficial lines and/or deep furrows on skin of the forehead.Crow's feet lines and wrinkles refer to superficial lines and/or deepfurrows on skin around the eye area. Marionette lines and wrinkles referto superficial lines and/or deep furrows on skin around the mouth.

It is also contemplated that the compositions of the invention will beuseful for treating thin skin by topically applying the composition tothin skin of an individual in need thereof. “Thin skin” is intended toinclude skin that is thinned due to chronological aging, menopause, orphoto-damage. In some embodiments, the treatment is for thin skin inmen, whereas other embodiments treat thin skin in women, pre-menopausalor post-menopausal, as it is believed that skin thins differently withage in men and women, and in particular in women at different stages oflife.

Without wishing to be bound by theory, it is believed that compoundsdescribed herein can act to increase paxillin levels in dermalfibroblasts, thereby maintaining cell shape and health, and delaying oneor more of the unwanted features associated with skin aging. It hassurprisingly been found that in human skin fibroblasts, paxillin proteinlevels decrease with aging. For example, dermal fibroblasts from olderdonors showed 20.6% lower paxillin levels compared to younger donors(n=3). Furthermore, interference of the normal production of paxillinmRNA and protein directly lead to changes in cell shape of human skinfibroblasts. Collapsed fibroblast have a lower rate of proliferation andreduced ability to produce collagen matrix Accordingly, increasingpaxillin levels can maintain youthful skin cell morphology and hencecellular function, including retarding cellular aging and stimulatingcollagen synthesis. More youthful paxillin levels may improve and/orrestore skin cell shape, increase cell proliferation and production ofextracellular matrix proteins, hence leading to overall healthier skinwith fewer lines and wrinkles

In certain preferred embodiments, the compositions and methods of theinstant invention are directed to improving skin firmness, plumpness,and/or tautness. Loss of firmness, wrinkling and other signs of agingresult in part from loss of skin collagen over time. As used herein“collagen” is used interchangeably with “collagen I” or “collagen typeI,” the type present in skin as a dermal matrix component. Collagen I iscomposed of three protein chains wound together in a tight triple helix,which provides a tensile strength greater than that of steel and iscreated by fibroblasts. Collagen gives skin firmness, strength,durability, and a youthful, smooth, plump appearance. Without wishing tobe bound by theory, it is believed increasing paxillin levels can leadto increased collagen production and thus collagen skin levels, therebydelaying one or more of the unwanted features associated with skinaging, e.g., by instead maintaining skin firmness and plumpness.

In certain embodiments, the compositions of the instant inventioncomprise a paxillin stimulator, such as cis-6-Nonenol, in an amountsufficient to increase paxillin level in a given area of skin whentopically applied thereto. As used herein, “increasing paxillin level”and related expressions refer to stimulating, inducing, or up-regulatingpaxillin mRNA (and protein) production to increase the paxillin contentin an area of skin, preferably improving skin appearance to aperceptible extent. For example, in some embodiments, the paxillin levelis increased by at least about 10%, at least about 20%, at least about60%, at least about 80%, at least about 100%, at least about 150%, atleast about 200%, at least about 250%, or at least about 300%, comparedto the level of paxillin in the absence of the composition. Paxillinlevels in the skin can be determined by appropriate assays, e.g., invitro assays described herein or known in the art. For example, Example1 below provides experimental details of assays for measuring paxillinlevels in human dermal fibroblasts.

In some embodiments, the cosmetic compositions for treating and/orpreventing signs of skin aging can further comprise additionalanti-aging agents. For example, the cosmetic composition comprising apaxillin stimulator (or paxillin stimulators) in an amount effective (oramounts effective) to treat and/or prevent signs of skin aging mayfurther comprise at least one other anti-aging agent. It is contemplatedthat synergistic improvements may be obtained with such combinations, insome embodiments.

Exemplary anti-aging agents include, without limitation, botanicals(e.g., Butea Frondosa extract); thiodipropionic acid (TDPA) and estersthereof; retinoids (e.g., all-trans retinoic acid, 9-cis retinoic acid,phytanic acid and others); hydroxy acids (including alpha-hydroxyacidsand beta-hydroxyacids), salicylic acid and salicylates; antioxidants,exfoliating agents (e.g., glycolic acid, 3,6,9-trioxaundecanedioic acid,etc.), estrogen synthetase stimulating compounds (e.g., caffeine andderivatives); compounds capable of inhibiting 5 alpha-reductase activity(e.g., linolenic acid, linoleic acid, finasteride, and mixturesthereof); barrier function enhancing agents (e.g., ceramides,glycerides, cholesterol and its esters, alpha-hydroxy and omega-hydroxyfatty acids and esters thereof, etc.); collagenase inhibitors; elastaseinhibitors; anti-aging botanicals, keratolytic agents, desquamatingagents, keratinocyte proliferation enhancers, and skin plumpers thatserve as additional collagen enhancers to the skin, to name a few. Anexample of a suitable skin plumper is palmitoyl oligopeptide. Other skinplumpers include other collagen and/or other glycosaminoglycan (GAG)enhancing agents. Exemplary retinoids include, without limitation,retinoic acid (e.g., all-trans or 13-cis) and derivatives thereof,retinol (Vitamin A) and esters thereof, such as retinol palmitate,retinol acetate and retinol propionate, and salts thereof. In someembodiments, the invention relates to synergistic action of one or morecompositions described herein with TDPA, e.g., to provide enhancedanti-aging benefits to skin.

Based on the teachings provided herein, one of skill in the art willrecognize other cosmetic and/or pharmaceutical applications for thecompositions described herein, and such applications are alsocontemplated as within the scope of the instant invention. For example,compositions described herein may also find use in personal careproducts, such as skin care products, where it is desirable to produce askin benefit described herein upon application of the product. Personalcare products for the skin include, for example, body lotions, bodywashes, body tonics, and the like. It is contemplated, for example, thatcompositions described herein can find use in lotion, tonic, and/or washformulations that decrease the appearance of lines and wrinkles onvarious surfaces of the body.

The paxillin stimulator is topically applied to an individual in needthereof, by which is meant an individual standing to benefit fromreducing visible signs of skin damage or aging. The invention providesmethods for providing a skin benefit by topically applying a compositioncomprising at least one paxillin stimulator over an area of skin for aperiod of time sufficient to produce one or more of the benefitsdescribed herein. The composition will typically be applied to the skinone, two, or three times daily for as long as is necessary to achievedesired results, such as anti-aging benefits described herein. Thistreatment regiment may comprise daily application or every-other-dayapplication for at least about one week, at least about two weeks, atleast about four weeks, at least about eight weeks, at least abouttwelve weeks, or more. Chronic treatment regimens are also contemplated,e.g., with respect to prophylactic treatments aimed at forestalling oneor more signs of skin aging or other damage. The treatment and/orprophylactic regime may also depend on specific the paxillinstimulator(s) being used, e.g., as certain paxillin stimulators mayproduce anti-aging skin benefits more quickly than others.

Additional candidate paxillin stimulators for use as anti-aging agentsmay be screened for use in treating skin in need thereof, e.g., asdescribed in more detail below.

Methods for Screening Candidate Paxillin Stimulators

Still another aspect of the instant invention relates to screeningcandidate paxillin stimulators, e.g., to find substances suitable forformulating an anti-aging cosmetic composition by incorporation into acosmetically acceptable vehicle. A vast array of substances can bescreened and it should be understood, although not always explicitlystated, that a candidate paxillin stimulator may be used alone or incombination with another paxillin stimulator.

In some embodiments, the candidate agents encompass natural, syntheticor semi-synthetic organic compounds based on various core structures.The agent may encompass one or more numerous chemical classes,preferably comprising functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, e.g.,typically including at least an amine, carbonyl, hydroxyl or carboxylgroup, and frequently at least two of the functional chemical groups.Candidate agents are also found among biomolecules including, but notlimited to peptides, saccharides, fatty acids, steroids, purines,pyrimidines, benzodiazapines, derivatives, structural analogs,polynucleotides, macromolecular complexes, or combinations thereof.

Candidate agents may be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. In someembodiments, the candidate paxillin stimulator may be any organic orinorganic compound and a number of natural and/or synthetic libraries ofcompounds can be used to provide candidate agents. See, e.g., NCl OpenSynthetic Compound Collection library, Bethesda, Md.; Pirrung et al.,2008, “Synthetic Libraries of Fungal Natural Products” ChemInform 39:2;Shang et al., 2005, “Advancing chemistry and biology throughdiversity-oriented synthesis of natural product-like libraries” Curr.Opin. Chem. Biol. 9:248-58; Webb TR, 2005, “Current directions in theevolution of compound libraries” Curr. Opin. Drug Discov. Devel.8:303-8; Fodor et al., 1991, Science 251:767-73; Medynski, 1994,BioTechnology 12:709-710; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci.USA 90:10922-26; Erb et al., 1994, Proc. Natl. Acad. Sci. USA91:11422-26; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA91:1614-18; and Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-712).

Further, numerous means are available for random and directed synthesisof a wide variety of organic compounds and biomolecules, includingexpression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc., to produce structural analogs. Candidate agents mayalso include cell lysates or sub-cellular compartments, extracts ofplant or animal origin, or any combinations thereof.

The candidate agent may be assayed to determine whether it can stimulatepaxillin by any means known in the art and/or described herein. Forexample, Example 1 below describes an assay for screening for candidatepaxillin stimulators by testing for increase in paxillin mRNA. In someembodiments, the method involves contacting a human skin cell with thecandidate agent or candidate material and assaying for up-regulation ofpaxillin mRNA. The concentration of candidate agent in a test samplewill vary depending on the nature of the agent. For example, thecandidate agent can be tested at one or more concentrations, e.g., atabout 5%, at about 2%, about 1%, 0.1%, about 0.05%, about 0.01%, about0.001%, about 0.0001%, about 0.00001%, about 0.000001%, and the like.

In some preferred embodiments, up-regulation of paxillin mRNA is assayedusing a multiplex assay that employs a branched DNA technology. Thistechnology is a hybridization-based method of target-specific RNAquantification that amplifies a signal(s) rather than target RNA, usinglabelled DNA probes. Probes for paxillin have been designed andsynthesized. The probes preferably cover the target sequence to beidentified. That is, the probes can be designed such that the nucleotidesequence corresponding to paxillin mRNA is covered or substantiallycovered by complementary sequences of the various probes. The probesgenerally include capture extenders (CE), label extenders (LE), andblockers (BL). Capture extenders are short sequences that bind the RNAto the beads used, e.g., beads commercially available from Luminex.Label extenders are short sequences that bind the RNA to DNA oligomersused in amplification (bDNA amplification oligos, which also may bereferred to as Premplifier, Amplifier, or Label Probes). Blockers areshort sequences that bind to RNA at regions within the target sequencethat are not covered by the CE or the LE. This arrangement provides atarget sequence that that is double-stranded, as the target sequence iscovered (or substantially covered) by the combination of various probesdirected to different regions of the target. Without wishing to be boundby theory, it is believed that providing a double-stranded targetsequence improves specificity and/or sensitivity of the assay, forexample, by improving hybridization efficiency.

In one embodiment, CE, LE, and BL sequences for identifying paxillincorrespond to SEQ ID NOS: 1, 2, and 3, respectively, as set forth below:

(SEQ ID NO: 1)gggaagacgtggcacccctcccggaacttcttcgagccccgcgctgctactactgcaacggccccatcctggaagccttctttggtcccgaaaacggcagcttcttcgagc; (SEQ ID NO: 2)gagcacttcgtctgcacccactgccaggaggagatcggataaagtggtgacagcccttgaccggacgtggcaccctgagggttccacgagaaggacggcaaggcctactgtcgcaagtgctttgtgtgccgggaatgcttcacgccattcgtgacgacgggcagccctactgtgaggtgcactaccacgagc; and (SEQ ID NO: 3)acacttcttctgtgcacagtgtggggactacttcgacatgttcgcacccaagtgtggcggctgcgcccgggccatcctggagaactatatctcagccctcaacacgctgtggcatcctga.

In other embodiments, the probes used may comprise one or morevariations from the above-recited sequences. For example, in someembodiments, the CE, LE, and/or BL probe has a nucleotide sequence thatis at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or at least about 99%identical to the nucleotide sequence of SEQ ID NOS: 1, 2, and 3,respectively. In some embodiments, the CE, LE, and/or BL probe has anucleotide sequence that hybridizes under stringent conditions to anucleotide sequence of SEQ ID NOS: 1, 2, and 3, respectively. Stringentconditions can include conditions of low stringency, moderatestringency, or high stringency.

“High stringency conditions” can include, but are not limited to, thosethat (1) employ low ionic strength and high temperature for washing, forexample 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C.; (2) employ, during hybridization, adenaturing agent, such as formamide, for example, 50% (v/v) formamidewith 0.1% bovine serum albumin/0.1% Ficol1/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mMsodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, SXDenhardt's solution, sonicated salmon sperm DNA(50 μg/mL), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at55° C., followed by a high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C. By way of example and not limitation,procedures using conditions of high stringency are as follows.Prehybridization of filters containing DNA is carried out for 8 h toovernight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. inprehybridization mixture containing 100 μg/ml denatured salmon sperm DNAand 5−20×10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minbefore autoradiography. Other conditions of high stringency which may beused are well known in the art. Selection of appropriate conditions forsuch stringencies is well known in the art (see e.g., Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; see also, Ausubel et al.,eds., in the Current Protocols in Molecular Biology series of laboratorytechnique manuals©, 1987-1997, Current Protocols©, 1994-1997 John Wileyand Sons, Inc.; see also, Dyson, 1991, “Immobilization of nucleic acidsand hybridization analysis,” In: Essential Molecular Biology: APractical Approach, Vol. 2, T. A. Brown, ed., pp. 111-156, IRL Press atOxford University Press, Oxford, UK) (each of which is herebyincorporated by reference herein in its entirety).

“Moderately stringent conditions” are described by, but not limited to,those in Sambrook et al., Molecular Cloning: A Laboratory Manual.2.sup.nd Ed., New York: Cold Spring Harbor Press, 1989, and include theuse of washing solution and hybridization conditions (e.g., temperature,ionic strength and % SDS) less stringent than those described above. Anexample of moderately stringent conditions is overnight incubation at37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.

“Low stringency conditions” can include, but are not limited to, thefollowing. Filters containing DNA are pretreated for 6 h at 40° C. in asolution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmonsperm DNA. Hybridizations are carried out in the same solution with thefollowing modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/mlsalmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5−20×10⁶ cpm³²P-labeled probe is used. Filters are incubated in hybridizationmixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. in asolution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS. The wash solution is replaced with fresh solution and incubated anadditional 1.5 h at 60° C. Filters are blotted dry and exposed forautoradiography. If necessary, filters are washed for a third time at65-68° C. and re-exposed to film. Other conditions of low stringencywhich may be used are well known in the art (e.g., as employed forcross-species hybridizations). (see also Shilo and Weinberg, 1981, Proc.Natl. Acad. Sci. U.S.A. 78, 6789-6792).

Assay methods using the labelled probes involve capturing the targetsequence and identifying captured sequences using one or more signalamplification steps. In some embodiments, appropriately diluted celllysates are hybridized with paxillin and reference probes. Once thepaxillin and the reference mRNAs are captured, the unbound material canfiltered and washed. Signal amplification can be performed in one ormore steps, typically in two steps using a “Pre-amplifier” probe,followed by an “Amplifier” probe, where unbound Pre-amplifier isfiltered and washed before amplification with the Amplifier. The samplesthen can be hybridized with a labelled probe, filtered and washed asbefore, and finally, the labels read or measured.

Increase in paxillin level may be measured as a percent increaserelative to a control. Preferred paxillin stimulators increase paxillinlevels by at least about 60%, at least about 80%, at least about 100%,at least about 150%, at least about 200%, at least about 250%, or atleast about 300%, compared to the level of paxillin in the absence ofthe composition or to that in the presence of a control. A candidatematerial thus can be identified as a paxillin stimulator, e.g., acandidate material can be identified as up-regulating paxillin mRNAusing one or more probes, compositions, and/or methods described herein,and such identified candidate materials are encompassed within the scopeof the instant invention.

Identified paxillin stimulators may be used to formulate cosmeticcompositions, as known in the art. The cosmetic compositions find use inanti-aging products, preferably formulated for topical application tothe skin e.g., with a cosmetically acceptable vehicle. Formulations foranti-aging cosmetic products comprising paxillin stimulators aredescribed in more detail below.

Cosmetic Formulations of Paxillin Stimulating Compounds

The compositions according to the invention can be formulated in avariety of forms for topical application and will comprise from about0.000001% to about 5% by weight of paxillin stimulator, from about0.00001% to about 2% by weight of paxillin stimulator, and preferablywill comprise from about 0.0001% to about 1% by weight, more preferablyfrom about 0.001% to about 0.1% by weight, and even more preferably0.01% to about 0.05% by weight based on the total weight of thecomposition. The above amounts refer to an “active amount” of thepaxillin stimulator, such as the amount of cis-6-nonenol. The term“active amount” refers to the amount of paxillin stimulator absentdiluent, solvent, carrier, filler or the like. The compositions willcomprise effective amount(s) of paxillin stimulator(s), by which isgenerally meant amount(s) sufficient to increase paxllin mRNA and/orprotein levels in given area of skin when topically applied thereto fora sufficient period of time.

In some embodiments, the cosmetic composition includes cis-6-Nonenol andoptionally at least one other paxillin stimulator. In some suchembodiments, the composition is essentially free of the trans-6-nonenolisomer or essentially free of nonenol isomers having the double bond inpositions other than the 6-position. By “essentially free of” is meantthat such other nonenol constituents will comprise less than 5% byweight of the total amount of nonenol, preferably, less than 2.5% byweight, and more preferably, less than 1% by weight. In otherembodiments, the compositions will be free of nonenols other thancis-6-Nonenol.

The composition may be formulated in a variety of product forms, suchas, for example, a lotion, cream, serum, spray, aerosol, cake, ointment,essence, gel, paste, patch, pencil, towelette, mask, stick, foam,elixir, concentrate, and the like, particularly for topicaladministration. Preferably the composition is formulated as a lotion,cream, ointment, or gel.

The compositions can include a cosmetically acceptable vehicle,encompassing any pharmaceutically, physiologically ordermatologically-acceptable vehicle, diluent or carrier. Such vehiclesmay take the form of any known in the art suitable for application toskin and may include water (e.g., deionized water); vegetable oils;mineral oils; esters such as octal palmitate, isopropyl myristate andisopropyl palmitate; ethers such as dicapryl ether and dimethylisosorbide; alcohols such as ethanol and isopropanol; fatty alcoholssuch as cetyl alcohol, cetearyl alcohol, stearyl alcohol and biphenylalcohol; isoparaffins such as isooctane, isododecane and is hexadecane;silicone oils such as cyclomethicone, dimethicone, dimethiconecross-polymer, polysiloxanes and their derivatives, preferablyorganomodified derivatives; hydrocarbon oils such as mineral oil,petrolatum, isoeicosane and polyisobutene; polyols such as propyleneglycol, glycerin, butylene glycol, pentylene glycol and hexylene glycol;waxes such as beeswax and botanical waxes; or any combinations ormixtures of the foregoing.

The vehicle may comprise an aqueous phase, an oil phase, an alcoholphase, a silicone phase or mixtures thereof. The cosmetically acceptablevehicle may also comprise an emulsion. Non-limiting examples of suitableemulsions include water-in-oil emulsions, oil-in-water emulsions,silicone-in-water emulsions, water-in-silicone emulsions, wax-in-wateremulsions, water-oil-water triple emulsions, or the like having theappearance of a cream, gel or microemulsions. The emulsion may includean emulsifier, such as a nonionic, anionic or amphoteric surfactant.

The oil phase of the emulsion preferably has one or more organiccompounds, including emollients; humectants (such as butylene glycol,propylene glycol, Methyl gluceth-20, and glycerin); otherwater-dispersible or water-soluble components including thickeners suchas Veegum or hydroxyalkyl cellulose; gelling agents, such as high MWpolyacrylic acid, i.e. CARBOPOL 934; and mixtures thereof. The emulsionmay have one or more emulsifiers capable of emulsifying the variouscomponents present in the composition.

The compounds suitable for use in the oil phase include withoutlimitation, vegetable oils; esters such as octyl palmitate, isopropylmyristate and isopropyl palmitate; ethers such as dicapryl ether; fattyalcohols such as cetyl alcohol, stearyl alcohol and behenyl alcohol;isoparaffins such as isooctane, isododecane and isohexadecane; siliconeoils such as dimethicones, cyclic silicones, and polysiloxanes;hydrocarbon oils such as mineral oil, petrolatum, isoeicosane andpolyisobutene; natural or synthetic waxes; and the like. Suitablehydrophobic hydrocarbon oils may be saturated or unsaturated, have analiphatic character and be straight or branched chained or containalicyclic or aromatic rings. The oil-containing phase may be composed ofa singular oil or mixtures of different oils.

Hydrocarbon oils include those having 6-20 carbon atoms, more preferably10-16 carbon atoms. Representative hydrocarbons include decane,dodecane, tetradecane, tridecane, and C₈₋₂₀ isoparaffins. Paraffinichydrocarbons are available from Exxon under the ISOPARS trademark, andfrom the Permethyl Corporation. In addition, C₈₋₂₀ paraffinichydrocarbons such as C₁₂ isoparaffin (isododecane) manufactured by thePermethyl Corporation having the tradename Permethyl 99A™ are alsocontemplated to be suitable. Various commercially available C₁₆isoparaffins, such as isohexadecane (having the tradename Permethyl®)are also suitable. Examples of preferred volatile hydrocarbons includepolydecanes such as isododecane and isodecane, including for example,Permethyl-99A (Presperse Inc.) and the C₇-C₈ through C₁₂-C₁₅isoparaffins such as the Isopar Series available from Exxon Chemicals. Arepresentative hydrocarbon solvent is isododecane.

The oil phase may comprise one or more waxes, including for example,rice bran wax, carnauba wax, ouricurry wax, candelilla wax, montanwaxes, sugar cane waxes, ozokerite, polyethylene waxes, Fischer-Tropschwaxes, beeswax, microcrystalline wax, silicone waxes, fluorinated waxes,and any combination thereof.

Non-limiting emulsifiers included emulsifying waxes, emulsifyingpolyhydric alcohols, polyether polyols, polyethers, mono- or di-ester ofpolyols, ethylene glycol mono-stearates, glycerin mono-stearates,glycerin di-stearates, silicone-containing emulsifiers, soya sterols,fatty alcohols such as cetyl alcohol, acrylates, fatty acids such asstearic acid, fatty acid salts, and mixtures thereof. The preferredemulsifiers include soya sterol, cetyl alcohol, stearic acid,emulsifying wax, acrylates, silicone containing emulsifiers and mixturesthereof. Other specific emulsifiers that can be used in the compositionof the present invention include, but are not limited to, one or more ofthe following: sorbitan esters; polyglyceryl-3-diisostearate; C₁₀₋₃₀alkyl acrylate crosspolymer; Dimethicone PEG-7 isostearate, acrylamidecopolymer; mineral oil; sorbitan monostearate, sorbitan tristearate,sorbitan sesquioleate, sorbitan monooleate; glycerol esters such asglycerol monostearate and glycerol monooleate; polyoxyethylene phenolssuch as polyoxyethylene octyl phenol and polyoxyethylene nonyl phenol;polyoxyethylene ethers such as polyoxyethylene cetyl ether andpolyoxyethylene stearyl ether; polyoxyethylene glycol esters;polyoxyethylene sorbitan esters; dimethicone copolyols; polyglycerylesters such as polyglyceryl-3-diisostearate; glyceryl laurate;Steareth-2, Steareth-10, and Steareth-20, to name a few. Additionalemulsifiers are provided in the INCI Ingredient Dictionary and Handbook11th Edition 2006, the disclosure of which is hereby incorporated byreference.

These emulsifiers typically will be present in the composition in anamount from about 0.001% to about 10% by weight, in particular in anamount from about 0.01% to about 5% by weight, and more preferably, fromabout 0.1% to about 3% by weight.

The oil phase may comprise one or more volatile and/or non-volatilesilicone oils. Volatile silicones include cyclic and linear volatiledimethylsiloxane silicones. In one embodiment, the volatile siliconesmay include cyclodimethicones, including tetramer (D4), pentamer (D5),and hexamer (D6) cyclomethicones, or mixtures thereof. Particularmention may be made of the volatile cyclomethicone-hexamethylcyclotrisiloxane, octamethyl-cyclotetrasiloxane, anddecamethyl-cyclopentasiloxane. Suitable dimethicones are available fromDow Corning under the name Dow Corning 200® Fluid and have viscositiesranging from 0.65 to 600,000 centistokes or higher. Suitable non-polar,volatile liquid silicone oils are disclosed in U.S. Pat. No. 4,781,917,herein incorporated by reference in its entirety. Additional volatilesilicones materials are described in Todd et al., “Volatile SiliconeFluids for Cosmetics”, Cosmetics and Toiletries, 91:27-32 (1976), hereinincorporated by reference in its entirety. Linear volatile siliconesgenerally have a viscosity of less than about 5 centistokes at 25° C.,whereas the cyclic silicones have viscosities of less than about 10centistokes at 25° C. Examples of volatile silicones of varyingviscosities include Dow Corning 200, Dow Corning 244, Dow Corning 245,Dow Corning 344, and Dow Corning 345, (Dow Corning Corp.); SF-1204 andSF-1202 Silicone Fluids (G.E. Silicones), GE 7207 and 7158 (GeneralElectric Co.); and SWS-03314 (SWS Silicones Corp.). Linear, volatilesilicones include low molecular weight polydimethylsiloxane compoundssuch as hexamethyldisiloxane, octamethyltrisiloxane,decamethyltetrasiloxane, and dodecamethylpentasiloxane, to name a few.

Non-volatile silicone oils will typically comprise polyalkylsiloxanes,polyarylsiloxanes, polyalkylarylsiloxanes, or mixtures thereof.Polydimethylsiloxanes are preferred non-volatile silicone oils. Thenon-volatile silicone oils will typically have a viscosity from about 10to about 60,000 centistokes at 25° C., preferably between about 10 andabout 10,000 centistokes, and more preferred still between about 10 andabout 500 centistokes; and a boiling point greater than 250° C. atatmospheric pressure. Non limiting examples include dimethylpolysiloxane (dimethicone), phenyl trimethicone, anddiphenyldimethicone. The volatile and non-volatile silicone oils mayoptionally be substituted will various functional groups such as alkyl,aryl, amine groups, vinyl, hydroxyl, haloalkyl groups, alkylaryl groups,and acrylate groups, to name a few.

The water-in-silicone emulsion may be emulsified with a nonionicsurfactant (emulsifier) such as, for example,polydiorganosiloxane-polyoxyalkylene block copolymers, including thosedescribed in U.S. Pat. No. 4,122,029, the disclosure of which is herebyincorporated by reference. These emulsifiers generally comprise apolydiorganosiloxane backbone, typically polydimethylsiloxane, havingside chains comprising -(EO)_(m)- and/or —(PO)_(n)— groups, where EO isethyleneoxy and PO is 1,2-propyleneoxy, the side chains being typicallycapped or terminated with hydrogen or lower alkyl groups (e.g., C₁₋₆,typically C₁₋₃). Other suitable water-in-silicone emulsifiers aredisclosed in U.S. Pat. No. 6,685,952, the disclosure of which is herebyincorporated by reference herein. Commercially availablewater-in-silicone emulsifiers include those available from Dow Corningunder the trade designations 3225C and 5225C FORMULATION AID; SILICONESF-1528 available from General Electric; ABIL EM 90 and EM 97, availablefrom Goldschmidt Chemical Corporation (Hopewell, Va.); and the SILWETseries of emulsifiers sold by OSI Specialties (Danbury, Conn.).

Examples of water-in-silicone emulsifiers include, but are not limitedto, dimethicone PEG 10/15 crosspolymer, dimethicone copolyol, cetyldimethicone copolyol, PEG-15 lauryl dimethicone crosspolymer,laurylmethicone crosspolymer, cyclomethicone and dimethicone copolyol,dimethicone copolyol (and) caprylic/capric triglycerides, polyglyceryl-4isostearate (and) cetyl dimethicone copolyol (and) hexyl laurate, anddimethicone copolyol (and) cyclopentasiloxane. Preferred examples ofwater-in-silicone emulsifiers include, without limitation, PEG/PPG-18/18dimethicone (trade name 5225C, Dow Corning), PEG/PPG-19/19 dimethicone(trade name BY25-337, Dow Corning), Cetyl PEG/PPG-10/1 dimethicone(trade name Abil EM-90, Goldschmidt Chemical Corporation), PEG-12dimethicone (trade name SF 1288, General Electric), lauryl PEG/PPG-18/18methicone (trade name 5200 FORMULATION AID, Dow Corning), PEG-12dimethicone crosspolymer (trade name 9010 and 9011 silicone elastomerblend, Dow Corning), PEG-10 dimethicone crosspolymer (trade name KSG-20,Shin-Etsu), dimethicone PEG-10/15 crosspolymer (trade name KSG-210,Shin-Etsu), and dimethicone PEG-7 isostearate.

The water-in-silicone emulsifiers typically will be present in thecomposition in an amount from about 0.001% to about 10% by weight, inparticular in an amount from about 0.01% to about 5% by weight, and morepreferably, below 1% by weight.

The aqueous phase of the emulsion may include one or more additionalsolvents, including lower alcohols, such as ethanol, isopropanol, andthe like. The volatile solvent may also be a cosmetically acceptableester such as butyl acetate or ethyl acetate; ketones such as acetone orethyl methyl ketone; or the like.

The oil-containing phase will typically comprise from about 10% to about99%, preferably from about 20% to about 85%, and more preferably fromabout 30% to about 70% by weight, based on the total weight of theemulsion, and the aqueous phase will typically comprise from about 1% toabout 90%, preferably from about 5% to about 70%, and more preferablyfrom about 20% to about 60% by weight of the total emulsion. The aqueousphase will typically comprise from about 25% to about 100%, moretypically from about 50% to about 95% by weight water.

In certain embodiments, the composition may comprise up to about 70% byweight of volatile solvent(s), including volatile organic solvents.Specifically, the composition may comprise up to about 60%, preferablyup to about 50%, more preferably up to about 40%, and even morepreferably up to about 30% by weight of volatile solvent(s). In otherembodiments, the composition may be free of volatile solvents, includingvolatile organic solvents.

The compositions may include liposomes. The liposomes may comprise otheradditives or substances and/or may be modified to more specificallyreach or remain at a site following administration.

The composition may optionally comprise other cosmetic actives andexcipients, obvious to those skilled in the art including, but notlimited to, fillers, emulsifying agents, antioxidants, surfactants, filmformers, chelating agents, gelling agents, thickeners, emollients,humectants, moisturizers, vitamins, minerals, viscosity and/or rheologymodifiers, sunscreens, keratolytics, depigmenting agents, retinoids,hormonal compounds, alpha-hydroxy acids, alpha-keto acids,anti-mycobacterial agents, antifungal agents, antimicrobials,antivirals, analgesics, lipidic compounds, anti-allergenic agents, H1 orH2 antihistamines, anti-inflammatory agents, anti-irritants,antineoplastics, immune system boosting agents, immune systemsuppressing agents, anti-acne agents, anesthetics, antiseptics, insectrepellents, skin cooling compounds, skin protectants, skin penetrationenhancers, exfollients, lubricants, fragrances, colorants, depigmentingagents, hypopigmenting agents, preservatives (e.g., DMDMHydantoin/Iodopropynylbutylcarbonate), stabilizers, pharmaceuticalagents, photostabilizing agents, neutralizers (e.g., triethanolamine)and mixtures thereof. In addition to the foregoing, the cosmeticcompositions of the invention may contain any other compound for thetreatment of skin disorders.

Colorants may include, for example, organic and inorganic pigments andpearlescent agents. Suitable inorganic pigments include, but are notlimited to, titanium oxide, zirconium oxide and cerium oxide, as well aszinc oxide, iron oxide, chromium oxide and ferric blue. Suitable organicpigments include barium, strontium, calcium, and aluminium lakes andcarbon black. Suitable pearlescent agents include mica coated withtitanium oxide, with iron oxide, or with natural pigment.

Various fillers and additional components may be added. Fillers arenormally present in an amount of about 0 weight % to about 20 weight %,based on the total weight of the composition, preferably about 0.1weight % to about 10 weight %. Suitable fillers include withoutlimitation silica, treated silica, talc, zinc stearate, mica, kaolin,Nylon powders such as Orgasol™, polyethylene powder, Teflon™, starch,boron nitride, copolymer microspheres such as Expancel™ (NobelIndustries), Polytrap™ (Dow Corning) and silicone resin microbeads(Tospearl™ from Toshiba), and the like.

In one embodiment of the invention, the compositions of the inventionmay include a fragrance. Fragrances are substances which can impart anaesthetically pleasing aroma to the composition. Typical fragrancesinclude aromatic materials extracted from botanical sources (i.e., rosepetals, gardenia blossoms, jasmine flowers, etc.) which can be usedalone or in any combination to create essential oils. Alternatively,alcoholic extracts may be prepared for compounding fragrances. However,due to the relatively high costs of obtaining fragrances from naturalsubstances, the modern trend is to use synthetically preparedfragrances, particularly in high-volume products. One or more fragrancescan optionally be included in the composition in an amount ranging fromabout 0.001 to about 5 weight percent, preferably about 0.01 to about0.5 percent by weight. Fragrance may also be imparted to the compositionby the paxillin stimulator, e.g., where the paxillin stimulatorcomprises a plant extract having a pleasant or desirable aroma. In otherembodiments, the compositions of the invention will be fragrance-free,by which is meant that the composition will not contain fragrances, inparticular components that are added for the primary benefit ofproviding aroma.

The present compositions may also contain one or more insect repellentactives. Such actives include, but are not limited to, N,Ndiethyl-m-toluamide (DEET), ethyl butylacetylaminopropionate (IR3535 byMerck Co.), hydroxyethyl isobutyl piperidine carboxylate (1-piperidinecarboxylic acid) (Bayer KBR 3023), p-menthane-3,8-diol, oil ofcitronella, soy bean oil, lemon grass oil, geranium/geraniol oil, neemoil and other natural essential oils, p-menthane-3,8-diol, or anymixtures thereof. The insect repellent active may be present in anamount about 0.05 wt % to about 90 wt %, preferably about 0.1 wt % toabout 50 wt %, and most preferably about 0.1 wt % to about 30 wt %,based on the total weight of the composition. In other embodiments, thecompositions of the invention will be free of an insect repellentactive, by which is meant that the composition will not contain insectrepellents, e.g., components that are typically added for the primarybenefit of repelling insects.

In one embodiment of the invention, the compositions may includeadditional skin actives such as, but are not limited to, botanicals,keratolytic agents, desquamating agents, keratinocyte proliferationenhancers, collagenase inhibitors, elastase inhibitors, depigmentingagents, anti-inflammatory agents, steroids, anti-acne agents,antioxidants, salicylic acid or salicylates, thiodipropionic acid oresters thereof, advanced glycation end-product (AGE) inhibitors andalpha-hydroxyacids.

In a specific embodiment, the composition may comprise at least oneadditional botanical, such as, for example, a botanical extract, anessential oil, or the plant itself. Suitable botanicals include, withoutlimitation, extracts from Abies pindrow, Acacia catechu, Anogeissuslatifolia, Asmunda japonica, Azadirachta indica, Butea frondosa, Buteamonosperma, Cedrus deodara, Emblica officinalis, Ficus benghalensis,Glycyrrhiza glabra, Ilex purpurea Hassk, Innula racemosa, Ligusticumchiangxiong, Ligusticum lucidum, Mallotus philippinensis, Mimusopselengi, Morinda citrifolia, Moringa oleifera, Naringi crenulata, Neriumindicum, Psoralea corylifolia, Stenoloma chusana, Terminalia bellerica,tomato glycolipid and mixtures thereof.

The composition may comprise additional active ingredients havinganti-aging benefits, as it is contemplated that synergistic improvementsmay be obtained with such combinations. Exemplary anti-aging componentsinclude, without limitation, botanicals (e.g., Butea Frondosa extract);thiodipropionic acid (TDPA) and esters thereof; retinoids (e.g.,all-trans retinoic acid, 9-cis retinoic acid, phytanic acid and others);hydroxy acids (including alpha-hydroxyacids and beta-hydroxyacids),salicylic acid and salicylates; exfoliating agents (e.g., glycolic acid,3,6,9-trioxaundecanedioic acid, etc.), estrogen synthetase stimulatingcompounds (e.g., caffeine and derivatives); compounds capable ofinhibiting 5 alpha-reductase activity (e.g., linolenic acid, linoleicacid, finasteride, and mixtures thereof); barrier function enhancingagents (e.g., ceramides, glycerides, cholesterol and its esters,alpha-hydroxy and omega-hydroxy fatty acids and esters thereof, etc.);collagenase inhibitors; and elastase inhibitors, to name a few.

Exemplary retinoids include, without limitation, retinoic acid (e.g.,all-trans or 13-cis) and derivatives thereof, retinol (Vitamin A) andesters thereof, such as retinol palmitate, retinol acetate and retinolpropionate, and salts thereof.

In another embodiment, the topical compositions of the present inventionmay also include one or more of the following: a skin penetrationenhancer, an emollient, a skin plumper, an optical diffuser, asunscreen, an exfoliating agent, and an antioxidant.

An emollient provides the functional benefits of enhancing skinsmoothness and reducing the appearance of fine lines and coarse wrinklesExamples include isopropyl myristate, petrolatum, isopropyl lanolate,silicones (e.g., methicone, dimethicone), oils, mineral oils, fatty acidesters, cetyl ethylhexanoate, C₁₂₋₁₅ alkyl benzoate, isopropylisostearate, diisopropyl dimer dillinoeate, or any mixtures thereof. Theemollient may be preferably present from about 0.1 wt % to about 50 wt %of the total weight of the composition.

A skin plumper serves as an additional collagen enhancer to the skin. Anexample of a suitable, and preferred, skin plumper is palmitoyloligopeptide. Other skin plumpers are collagen and/or otherglycosaminoglycan (GAG) enhancing agents. When present, the skin plumpermay comprise from about 0.1 wt % to about 20 wt % of the total weight ofthe composition.

An optical diffuser is a particle that changes the surface optometricsof skin, resulting in a visual blurring and softening of, for example,lines and wrinkles Examples of optical diffusers that can be used in thepresent invention include, but are not limited to, boron nitride, mica,nylon, polymethylmethacrylate (PMMA), polyurethane powder, sericite,silica, silicone powder, talc, Teflon, titanium dioxide, zinc oxide, orany mixtures thereof. When present, the optical diffuser may be presentfrom about 0.01 wt % to about 20 wt % of the total weight of thecomposition.

A sunscreen for protecting the skin from damaging ultraviolet rays mayalso be included. Preferred sunscreens are those with a broad range ofUVB and UVA protection, such as octocrylene, avobenzone (Parsol 1789),octyl methoxycinnamate, octyl salicylate, oxybenzone, homosylate,benzophenone, camphor derivatives, zinc oxide, and titanium dioxide.When present, the sunscreen may comprise from about 0.01 wt % to about70 wt % of the composition.

Suitable exfoliating agents include, for example, alpha-hydroxyacids,beta-hydroxyacids, oxaacids, oxadiacids, and their derivatives such asesters, anhydrides and salts thereof. Suitable hydroxy acids include,for example, glycolic acid, lactic acid, malic acid, tartaric acid,citric acid, 2-hydroxyalkanoic acid, mandelic acid, salicylic acid andderivatives thereof. A preferred exfoliating agent is glycolic acid.When present, the exfoliating agent may comprise from about 0.1 wt % toabout 80 wt % of the composition.

An antioxidant functions, among other things, to scavenge free radicalsfrom skin to protect the skin from environmental aggressors. Examples ofantioxidants that may be used in the present compositions includecompounds having phenolic hydroxy functions, such as ascorbic acid andits derivatives/esters; alpha-hydroxyacids; beta-carotene; catechins;curcumin; ferulic acid derivatives (e.g., ethyl ferulate, sodiumferulate); gallic acid derivatives (e.g., propyl gallate); lycopene;reductic acid; rosmarinic acid; tannic acid; tetrahydrocurcumin;tocopherol and its derivatives (e.g., tocopheryl acetate); uric acid; orany mixtures thereof. Other suitable antioxidants are those that haveone or more thiol functions (—SH), in either reduced or non-reducedform, such as glutathione, lipoic acid, thioglycolic acid, and othersulfhydryl compounds. The antioxidant may be inorganic, such asbisulfites, metabisulfites, sulfites, or other inorganic salts and acidscontaining sulfur. Compositions of the present invention may comprise anantioxidant preferably from about 0.001 wt % to about 10 wt %, and morepreferably from about 0.01 wt % to about 5 wt %, of the total weight ofthe composition.

Other conventional additives include: vitamins, such as tocopherol andascorbic acid; vitamin derivatives such as ascorbyl monopalmitate;thickeners such as hydroxyalkyl cellulose; gelling agents; structuringagents such as bentonite, smectite, magnesium aluminum silicate andlithium magnesium silicate; metal chelating agents such as EDTA;pigments such as zinc oxide and titanium dioxide; colorants; emollients;and humectants.

It is preferred that the composition be essentially free of componentshaving a strong oxidizing potential, including for example, organic orinorganic peroxides. By “essentially free of” these components is meantthat the amounts present are insufficient to have a measurable impact onthe beneficial activity of the paxillin stimulator(s). In someembodiments, this will be on a molar basis in relation to the amount ofthe paxillin stimulator(s), less than 1%.

In one embodiment, the composition of the invention comprising at leastone paxillin stimulator may have a pH between about 1 and about 8. Incertain embodiments, the pH of the composition will be acidic, i.e.,less than 7.0., and preferably will be between about 2 and about 7, morepreferably between about 3.5 and about 5.5.

All terms used herein are intended to have their ordinary meaning unlessotherwise provided.

As used herein, “% by weight” or “% wt” refers to the weight percent ofa component in relation to the total weight of the composition (i.e.,including any carriers, vehicles, solvents, emollients, fillers, orother components added before application to the skin) unless otherwisespecified.

EXAMPLES Example 1 Stimulation of Paxillin mRNA In Vitro

Normal human dermal fibroblasts (Cascade Biologics) were cultured in96-well tissue culture treated plates, with 200 μl DMEM in 10% serum perwell, and incubated for 24 hours at 37° C. and 10% CO₂. In some cases,normal human epidermal keratinocytes (Cascade Biologics) were culturedin 200 μl EpiLife medium (Cascade Biologics) per well, and incubatedovernight at 37° C. and 5% CO₂.

Stock solutions of candidate paxillin stimulators (test materials) weremade in appropriate solvents (e.g., DMSO, water, ethanol, a 50:50ethanol:water mixture) to give weight % as indicated in Table 1 below.Cells were treated with test material or a respective vehicle controldiluted in growth medium for 24 hours in a humidified 37° C. incubatorwith 10% CO₂. After incubation, growth medium from each plate wasremoved and 1000 of lysis buffer was added to the wells and placed in37° C. incubator with 5% CO₂ for 30 minutes. At the end of incubation,the cells were collected in freezer plates and placed in a −80° C.freezer until analysis.

Changes in mRNA for paxillin in cell lysates after treatment wereanalysed using QuantiGene® multiplex assay (Panomics Inc. CA) thatemploys a branched DNA technology. This technology is ahybridization-based method of target-specific RNA quantification thatamplifies a signal(s) rather than target RNA, using labelled DNA probe.Probes for Paxillin gene (ID: NM 005953) were designed and synthesized.Paxillin probes, reference probes PPIB and GAPDH, along with a set ofother probes were used in this multiplex assay.

The probes used include capture extenders (CE), label extenders (LE),and blockers (BL). As described above, this arrangement provides atarget sequence that is double-stranded, thereby improving hybridizationefficiency. The CE, LE, and BL sequences used for identifying paxillinmRNA corresponded to SEQ ID NOS: 1, 2, and 3, respectively.

Appropriately diluted cell lysates were hybridized with the paxillin andreference probes in a hybridization plate in a Vortemp shakingincubator, for 18-22 hours at 54° C.±1° C. and 600 rpm. Once thepaxillin and the reference mRNAs were captured, the unbound material wasfiltered using filter plates and washed three times with a wash buffer.Signal amplification was performed using two steps—first a 2.0Pre-amplifier (Panomics Inc.) was added and incubated for 1 hour in theVortemp shaking incubator at 54° C.±1° C. and 600 rpm. UnboundPre-amplifier was filtered using a Filter plate and washed twice. Nextthe samples were exposed to 2.0 Amplifier (Panomics Inc.) and incubated,filtered, and washed as with the Pre-amplifier. Next the samples werehybridized with biotinylated labelled probe, and incubated for one hourat 54° C.±1° C. and 600 rpm. Samples were then filtered and washed asabove. Finally, Streptavidin-conjugated Phycoerythrin (SAPE) workingreagent was added and the mixture shaken for 30 minutes at roomtemperature covered in aluminium foil. Unbound SAPE was filtered andwashed twice. SAPE wash buffer was added to each well with shaking for2-5 minutes, and then readings were taken immediately using a Luminexmachine.

Values for amounts of paxillin mRNA were determined along with those forthe reference genes PPIB and GAPDH. Values were normalized to GAPDH todetermine changes in paxillin mRNA after treatment. Percent increase inmRNA for paxillin was calculated in each case by comparing values aftertreatment with a candidate paxillin stimulator to values after treatmentwith the vehicle control.

Results for exemplary paxillin stimulators are presented below in Table1.

TABLE 1 % increase in Paxillin (relative to Paxillin Stimulator Weight %control) Compound of Formula IV 0.00005%   31.67% Compound of Formula V0.00005%   28.52% Jasminium sambac extract 0.001%  73.77% Cocciniagrandis extract 0.10% 73.12% Eliptica prostrata Linn. extract 0.10%102.2% Clitoria ternatea Linn. extract 0.10%  54.0% 0.01%  57.1%Ozothamnus obcordatus extract 0.01% 21.55% Erythrina flabelliformisextract 0.001%  33.23% Lonchocarpus capassa extract 0.01% 24.94% Sophoratomentosa extract 0.01% 136.13%  Trifolium hybridum extract 0.01%239.06%  Eremophila mitchelli extract 0.01% 138.34%  Kunzea ambiguaextract 1 0.01% 132.51%  Kunzea ambigua extract 2 0.001%  250.88% Tanshinone IIA 0.025%  188.31%  Tetrandine 0.001%  84.31% Carvacrol0.0001%  39.50% cis-6-Noneol 0.0010%  60.84% Retinyl punicate 0.01%69.68% Retinyl oleate 0.01% 60.21% Equol 0.001%  220.36%  0.0001% 61.46% MycoFusions Coriolus Black 0.10% 27.55% Corn Biomass MycoFusionsMaitake Waxy Hulless 0.10% 24.28% Barley Biomass Zanthoxylum nitidiumextract 0.10% 73.28% Ophiopogon Thunb. PE extract 0.01% 115.21%  Radixplatycodonis extract 0.10% 68.92% Terminalia belerica extract 0.01%260.09%  Cocculus glaucescens 0.010%  36.32% Stephania solid extract0.10% 104.08%  Rosemary PE 50% extract 0.001%  172.75% 

Fibroblasts treated with the indicated weight % of the variousrespective paxillin stimulators showed a significant % stimulation inmRNA levels, as indicated in Table 1.

Example 2 Stimulation of Paxillin Protein in Human Skin Biopsy

Compositions comprising a paxillin stimulator were each tested on 21subject volunteers. For each paxillin stimulator, the composition wastopically applied via a patch attached to the back of a randomlyselected forearm of each of 21 volunteers, and a vehicle control wassimilarly applied to the back of the volunteer's forearm. After 3 weeks,the human skin biopsies were taken from the treated skin of the variousvolunteers and subjected to immunohistochemisty preparation. Paxillinprotein level was demonstrated by specific antibody stain followed bymicroscopic examinations. Elevated protein level was determined for eachtreated subject by comparing to the vehicle control. Results wereexpressed as percentage of subjects that showed improvement.

Results for exemplary paxillin stimulators are presented in Table 2.

TABLE 2 Paxillin Total No. of No. Showing % Subjects Stimulator SubjectsImprovement Improved cis-6-Nonenol (%) 21 15 71.4 Lonchocarpus 21¹ 630.0 capassa extract (%) ¹Results were not able to be read for onesubject treated with the topical composition comprising Lonchocarpuscapassa extract.

As illustrated in Table 2, cis-6-nonenol showed increased paxillinlevels in 71.4% of subjects tested; while Lonchocarpus capassa extractshowed positive results in 30.0%.

Further, increase in paxillin protein was visibly observed in human skinbiopsies. Representative photographs were taken from biopsy describedabove. FIGS. 1A and B demonstrate the results, where darker stainingrepresent paxillin protein. As FIG. 1 illustrates, treatment withcis-6-Nonenol (B) increased paxillin protein in human skin as comparedto treatment with a control (A).

Example 3 Exemplary Compositions

Cosmetic compositions comprising the paxillin stimulator cis-6-Nonenolfor topical application to the skin are provided in Table 3 below.

TABLE 3 Composition: 1 2 3 4 Components Weight % paxillin stimulator0.0005 0.001 0.0015 0.002 (cis-6-Nonenol) Acrylates/C10-30 Alkyl 1 1 1 1Acrylate Crosspolymer Cetyl Ethylhexanoate 10 10 10 10 C12-15 AlkylBenzoate 3.9 3.9 3.9 3.9 Isopropyl Isostearate 3 3 3 3 Diisopropyl dimerdillinoleate 0.1 0.1 0.1 0.1 Tocopheryl acetate 0.5 0.5 0.5 0.5 Butyleneglycol 2 2 2 2 Propylene glycol 1 1 1 1 Dimethicone PEG-7 0.5 0.5 0.50.5 isostearate Methyl gluceth-20 0.5 0.5 0.5 0.5 Triethanolamine 1 1 11 Acrylates/acrylamide 1.5 1.5 1.5 1.5 copolymer/mineral oil DMDMHydantoin/ 0.4 0.4 0.4 0.4 Iodopropynylbutylcarbonate Deionized waterq.s. q.s. q.s. q.s. Total: 100 100 100 100

All references including patent applications and publications citedherein are incorporated herein by reference in their entirety and forall purposes to the same extent as if each individual publication orpatent or patent application was specifically and individually indicatedto be incorporated by reference in its entirety for all purposes. Manymodifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

The invention claimed is:
 1. An emulsion for topical applicationcomprising an extract of Cocculus glaucescens vine.